Project Summary Little is known about antigen (Ag) specific memory B (BMEM) cells, in the context of a public health relevant pathogen. Our studies clearly resolve the subsets of non-circulating, influenza specific, resident memory B cells (BRM) in the lung. BRM establishment relied on early CD40 signaling and maintained a CD73 (ectoenzyme)+ and CD73- population, whereas the lymph node (LN) showed a decline in the CD73- population. This raised the question of the inherent differences in formation and function of CD73+ and CD73- BRMs. CD73 is a marker of germinal center (GC) emigrants in the LN and memory cells in the LN reside in niches close to macrophages that capture and transfer antigen and T cells which can provide help; therefore, we hypothesized that the CD73+BRMs will home to areas of organized lymphoid structures in the lung and be dependent on T cell help. Our RNASeq data shows NLRP3 (NOD-like receptor protein 3), an innate danger sensing complex, to be the most upregulated gene in CD73+BRMs. To our knowledge NLRP3 activity is not associated with B cell differentiation, which makes this finding novel and may inaugurate studies into innate like functions in B cells during adaptive responses. NLRP3 activity produces interleukin B (IL-1B) and IL-1B is a well-known up regulator of interleukin 6 (IL-6), which promotes GCs and plasma cell differentiation. As our CD73+BRMs may have higher NLRP3 expression, we hypothesize that NLRP3 will be active during recall and produce GCs and ASCs, while the CD73-BRMs may be sentinels in the lung which spontaneously convert to ASCs, in response to the returning virus. To test these hypotheses, we ask the following: 1) Are CD73+ and CD73- BRM cells dependent on GCs? If so, GC-specific blockade should reduce CD73+ BRMs in the lung and produce low affinity BCRs. 2) Do CD73+ BRMs preferentially reside in iBALT? If so, using histology, we should observe them congregating in the iBALT while the CD73-BRMs are scattered in the parenchyma. 3) Do CD73+ and CD73- BRM cells respond differently in functional assays? We will answer this question by performing in vitro assays for BRM differentiation to ASCs, using cytokine cocktails and sorted T cells. 4) Does the NLRP3 complex become activated in BRMs upon viral challenge? If so, using NLRP3 inflammasome reporter mice, we expect that CD73+ BRM cells, but not CD73- BRM cells to increase NLRP3 activity. 5) Does NLRP3 regulate the formation or function of BRMs? If so, B cell specific NLRP3 deficiency should alter the proportions of CD73+ and CD73-BRMs and their protective function with observable differences in morbidity and mortality. The findings from this study despite the outcome will be extremely important to allow for the resolution of a target population and potential molecular targets for an effective vaccine design.